Project Summary/Abstract The vitamin K cycle enzymes: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and vitamin K reductase (VKR) are responsible for the post-translational carboxylation of vitamin K-dependent (VKD) proteins into their biologically active forms. Carboxylation is mainly associated with blood coagulation, as four coagulation factors (factors II, VII, IX, and X) and three anticoagulant proteins (proteins C, S, and Z) require carboxylation for their function. With the discovery of new VKD proteins and their new biological functions, the importance of carboxylation has been expanded to vascular calcification, bone development, glucose metabolism, and cell proliferation. GGCX is the enzyme that directly modifies VKD proteins to their functional forms. Genetic variations in GGCX have been identified in patients with vitamin K-related disorders. However, it is not clear why some GGCX mutations cause bleeding disorders while other mutations result in non-bleeding symptoms. Additionally, one GGCX mutation can sometimes cause two distinct clinical phenotypes. However, it remains unclear as to why the administration of vitamin K can ameliorate one symptom but not the other. VKOR is a regulatory enzyme of the vitamin K cycle and the target of the oral anticoagulant, warfarin. The mechanism of VKOR's active site regeneration remains elusive. Recent studies have shown that superwarfarin poisonings (an anticoagulant more powerful than warfarin) are a growing public health concern and that vitamin K therapy for that is costly and inefficient. The enzyme that reduces vitamin K to its hydroquinone form to support VKD carboxylation is VKR. Despite decades of effort, the identity of VKR is still unknown. Our long-term research goal is to understand in detail the structure and function relationship of all vitamin K cycle enzymes within their native milieu for better control coagulation and other related physiological processes. The current proposal aims to solve the main questions remaining in the field (as mentioned above). To accomplish these goals, we propose the following specific aims: Specific Aim 1 - we will use our recently established CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 knockout reporter cell lines to study the effect of all currently identified naturally occurring GGCX mutations on the carboxylation of different VKD proteins associated with distinct clinical phenotypes; Specific Aim 2 - we will apply genetic code expansion technology to clarify VKOR's active site regeneration and design and synthesize novel vitamin K derivatives to rescue superwarfarin poisonings; and Specific Aim 3 - we will use the genome-wide CRISPR-Cas9 knockout library to screen for the unknown enzyme, VKR. We expect that information derived from these studies will help us understand how the three vitamin K cycle enzymes contribute to the complex mechanisms of carboxylation, thereby gaining new therapeutic insights into the control of blood coagulation and vascular calcification and improving therapies for vitamin K-related disorders.